Cytology is a branch of biology dealing with the study of the formation, structure, and function of cells. As applied in a laboratory setting, cytologists, cytotechnologists, and other medical professionals make medical diagnoses of a patient's condition based on visual examination of a sample of the patient's cells. Cytological techniques lend themselves well to the detection of abnormal cells and disease in the human body.
Cytological techniques are widely employed, because collection of cell samples for analysis is generally less invasive than traditional surgical pathological procedures such as biopsies, whereby a tissue sample is excised from the patient using specialized biopsy needles having spring loaded translatable stylets, fixed cannulae, and the like. Cell samples may be obtained from the patient by a variety of techniques including, for example, by scraping or swabbing an area, or by using a needle to aspirate body fluids from the chest cavity, bladder, spinal canal, or other appropriate area. The cell samples are subsequently transferred to a glass slide for viewing under magnification. Fixative and staining solutions may be applied to the cells on the glass slide for preserving the sample for archival purposes and for facilitating examination.
A typical cytological technique is a Papanicolaou (“Pap”) smear test in which cells are scraped from a woman's cervix and analyzed in order to detect the presence of abnormal cells—a precursor to the onset of cervical cancer. In a conventional Pap smear test, the cells are smeared onto a glass slide at the physician's office, and the labeled slide is then sent off to a laboratory for analysis under a microscope by a cytotechnician. More recently, liquid-based cytological techniques have been employed, which offer new ways of collecting, storing, and analyzing the cervical cells collected during a Pap smear. Instead of smearing the cells onto a microscope slide, the physician places the collected material into a vial filled with a liquid preservation medium, such as PreservCyt® transport medium, and sends the vial capped and labeled vial to a laboratory for slide preparation. During slide preparation, any unwanted material, such as bacteria, blood, mucus, or other debris, is filtered from the sample, and the remaining cells are placed onto a microscope slide as a monolayer specimen for review by a cytotechnician. In this manner, the cells are able to be more readily discerned and counted to ensure that an adequate number of cells have been evaluated. Oftentimes, slide preparation using liquid-based cytological samples is performed automatically—up to eighty slides at one time. During this automated procedure, air pressure may be used to manipulated the sample. In particular, suction is used to draw the fluid onto a cylindrical filter, and a blast of air is to transfer the cells from the filter onto the slide to create the specimen.
Whether a conventional or a fluid-based Pap smear is performed, the specimen will be classified as either normal or abnormal based on the microscopic analysis of the slide. An abnormal sample can be classified into one of the major categories defined by The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnosis, which categories include Low-Grade Squamous Intraepithelial Lesions (LSIL), High-Grade Squamous Intraepithelial Lesions (HSIL), Squamous Cell Carcinoma, Adenocarcinoma, Atypical Glandular cells of Undetermined Significance (AGUS), Adenocarcinoma in situ (AIS), and Atypical Squamous Cell (ASC), which can be further sub-divided into Atypical Squamous Cell, cannot exclude HSIL (ASC-H) and Atypical Squamous Cell of Undetermined Significance (ASC-US).
If a specimen is not classified as abnormal after analysis, the Pap smear is considered normal and the patient is returned to a routine pap screen schedule (typically, once a year). With the exception of ASC-US, if a specimen is classified into any one of these abnormal categories, the patient must undergo an inconvenient and relatively painful colposcopy and biopsy to further diagnose the presence of cancer or precancer (dysplasia). Until recently, if a specimen was classified as ASC-US, the patient had to undergo repeated Pap smears every few months in hopes of determining the nature of the abnormal cells, i.e., whether they might develop into cancerous lesions or clear up on their own. In some cases, a colposcopy and biopsy was performed.
Since 2000, a specific Human Papilloma Virus (HPV) deoxynucleic acid (DNA) test, referred to as the Hybrid Capture II HPV DNA assay, manufactured by Digene Corporation, has been used to determine whether patients, whose Pap smears have been classified as ASC-US, have HPV, the high-risk serotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) of which have been recognized as a necessary factor for the development of cervical cancer (over ninety percent of women with cervical cancer have high-risk HPV).
Based on the strong correlation between HPV and cervical cancer, it has been recommended that HPV DNA testing be used as a triage test for patients whose Pap smear results are classified as ASC-US. That is, patients who have ASC-US Pap smear results and negative HPV test results can be reassured that their short term risk of developing cervical cancer is very low, and that they could safely return to a routine Pap smear schedule. In contrast, women who have ASC-US Pap smear results and positive results for high-risk HPV should undergo the colposcopy/biopsy process in the same manner as if the Pap smear would have originally been classified as LSIL, HSIL, or any of the other abnormal categories. A recent study has shown that performing an HPV DNA test would ultimately spare forty to sixty percent of patients from undergoing a colposcopic/biopsy examination.
In the case where a conventional Pap smear has been performed, the patient will typically have to make another visit to the physician to obtain another sample for HPV DNA testing. In the case where a liquid-based Pap smear has been performed, however, the same sample used to perform the Pap smear analysis can be conveniently used to perform a “reflexive” HPV DNA test, thereby obviating the need for a repeat clinic visit and Pap smear. In this case, if a slide is positive for ASC-US, an aliquot (e.g., 4 ml) of the fluid sample is removed from the stored vial and sent to a molecular diagnostic laboratory for HPV DNA testing.
Theoretically, an HPV DNA test can be performed at any time during the Pap smear process. However, because it is currently not commercially practical to perform the HPV DNA test in conjunction with every Pap smear, the HPV DNA test is typically performed in response to a ASC-US Pap smear result. Thus, in the case of liquid-based Pap smears, the aliquot required for HPV DNA testing would have to be taken from the sample after being processed for slide preparation, which may potentially lead to molecular contamination issues, and in the case of automated Pap smear processing, aerosol cross-contamination resulting from the use of air pressure to manipulate the sample or liquid contamination resulting from the transfer of filtered solution found in the plumbing of the automated processor. Molecular contamination is not a trivial issue because the transfer of just one nucleic molecule can produce a false-positive for HPV.
Significantly, laboratories that perform HPV DNA tests are weary of molecular contamination—a well-known problem in molecular diagnostic laboratories, which typically employ special engineering and design features, laboratory practices, and monitoring activities to minimize molecular contamination in all of their activities. Because automated systems for making Pap smear slides are designed to safely and effectively handle cells—not nucleic acids, the sterilization protocols followed by technicians when operating these systems do not satisfy the strict molecular contamination control safeguards and procedures required by molecular diagnostic laboratories. Thus, due to the risk of cross-contamination, molecular diagnostic laboratories may not accept aliquots that have been taken from an already processed liquid-based Pap smear for fear of unnecessarily generating false HPV positives.
Besides being used for HPV DNA testing, aliquots from liquid-based Pap smear samples can also be used in DNA testing for other sexually transmitted diseases, such as Chlamydia trachomatis and Neisseria gonorrhoeae. However, false positives are a special problem when testing for Chlamydia trachomatis and Neisseria gonorrhoeae, because they could have enormous family and social repercussions. Thus, molecular diagnostic laboratories are even more reluctant to accept aliquots from already processed liquid-based Pap smear samples. Because testing for other sexually transmitted diseases need not be used to triage ASC-US specimens, and are intended to be performed in parallel to the Pap smear tests at the request of the physician, aliquots may be taken from the Pap smear samples prior to processing, e.g., by manually pipetting the aliquot from the vial, thereby minimizing the risk of cross-contamination. However, this step may still not satisfy the strict contamination prevention requirements imposed by molecular diagnostic laboratories.
Besides contamination issues, the pipetting of an aliquot from a fluid-based Pap smear sample, whether done before or after the sample is processed, and whether done for HPV testing or testing of any other sexually transmitted disease, increases cost in the form of manual labor (which involves not only pipetting the aliquot into an extra vial, but also labeling the vial), material costs for the pipettes and extra vials, and storage space, which when multiplied by the thousands of samples processed, becomes significant.
There thus is a need to provide an improved method and apparatus for obtaining an aliquot from a fluid-based cytological sample, such as a Pap smear sample.